Two major goals of drug metabolism studies are to identify pathways of biotransformation which lead to toxic or active metabolites, and to promote the formation of the least toxic metabolites by either altering the structure of the drug or administering a second agent which selectivily enhances a non-toxic pathway. In this investigation, the widely prescribed analgesic, acetaminophen, will be used to examine the effects of modifying structure on hepatotoxicity caused by this agent, and to probe mechanisms involved in reactive metabolite formation and hepatocellular injury. Because the rolf o N-hydroxylation in the hepatotoxicity and renal toxicity caused by acetaminophen and its O-ethylated analog, phenacetin, is unclear, we will develop a specific and sensitive assay for N-hydroxiacetaminophen based on gas chromatography-mass spectrometry and selected-ion monitoring. This assay will be used to probe the possible formation of N-hydroxyacetaminophen from acetaminophen and phenacetin in vivo, and to determine if there is any relationship between its formation and toxicity to the animal. High pressure liquid chromatography with electrochemical detection will be used to investigate the potential role of quionones in the formation of reactive metabolites of acetaminophen and methylated analogs of acetaminophen. In addition this technique will provide a sensitive assay for catachol metabolites of acetaminophen so that their contribution to the elaboration of acetaminophen toxicity can be assessed pharmaco-kinetically. Analogs of acetaminophen specifically labeled with deuterium will be used to probe a possible role for arene oxides in the formation of reactive acetaminophen metabolites. Specific tritium and carbon-14 labeling will also be carried out on acetaminophen, phenacetin, and a non-hepatotoxic regioisomer of acetaminophen, 3-hydroxyacetanilide, to better compare the nature of the covalent binding interaction of these structural and toxic variants of acetaminophen. Finally, a ring-methylated analog of acetaminophen, 2-methylacetaminophen, which is known to be active as an analgesic, will be tested in the animal models developed for acetaminophen toxicity to determine if it is less toxic than acetaminophen.